IL-12 is a pivotal cytokine that links the innate and adaptive immune responses. TNF-alpha also plays a key role in orchestrating inflammation and immunity. The reciprocal influence of these two inflammatory mediators on each other may have significant impact on the cytokine balance that shapes the type and extent of immune responses. To investigate the relationship between TNF-alpha and IL-12 production, we analyzed the effects of exposure of human monocyte-derived macrophages to TNF-alpha on LPS- or Staphylococcus aureus-induced IL-12 production in the presence or absence of IFN-gamma. TNF-alpha is a potent inhibitor of IL-12 p40 and p70 secretion from human macrophages induced by LPS or S. aureus. IL-10 is not responsible for the TNF-alpha-mediated inhibition of IL-12. TNF-alpha selectively inhibits IL-12 p40 steady-state mRNA, but not those of IL-12 p35, IL-1alpha, IL-1beta, or IL-6. Nuclear run-on analysis identified this specific inhibitory effect at the transcriptional level for IL-12 p40 without down-regulation of the IL-12 p35 gene. The major transcriptional factors identified to be involved in the regulation of IL-12 p40 gene expression by LPS and IFN-gamma, i.e., c-Rel, NF-kappaB p50 and p65, IFN regulatory factor-1, and ets-2, were not affected by TNF-alpha when examined by nuclear translocation and DNA binding. These data demonstrate a selective negative regulation on IL-12 by TNF-alpha, identifying a direct negative feedback mechanism for inflammation-induced suppression of IL-12 gene expression.